The goals of the original grant, titled "Noninvasive measurement of drug concentrations in tissue," have been successfully achieved. These entailed the development of the method of Optical Pharmacokinetics (OP) for noninvasive measurement in real time, and with site-specificity, of the concentrations of drugs or other compounds in tissue, and the demostration of the technology in pharmacokinetic studies of new agents with animal tumor models. Site-specific pharmacokinetics is important for chemotherapy, and especially for photodynamic therapy, since it is the difference between the dose-response curves of tumor tissue and normal tissue that must be exploited during treatment. By extending the OP method to rapid sequences of measurements, other areas of medical research are enabled. The goal of this Competing Renewal grant is to advance the OP technology and apply it to the assessment of angiogenesis and of the response to treatment by anti-angiogenic agents. OP measurements of the first-pass kinetics of optical contrast agents, administered in a short bolus, will be used to assess microvascular density. Further OP measurements over the subsequent tens of minutes will permit assessment of the leakage into the extracellular fluid space and evaluation of microvessel permeability (hence, assessing capillary integrity) in regions of cancer, with the goal of a noninvasive method to assess angiogenesis and response to anti-angiogenic treatment. The ability of the OP system to make a burst of many measurements within seconds will also permit evaluation of the biologic noise in concentration measurements. It is a goal to demonstrate that the OP method, in the case of superficial lesions, is able to provide information that is not available through CT and MRI techniques, and by use of a small, portable, and inexpensive device. We will design and assemble a next-generation OP system capable of >3 measurements per second, for determining local agent concentrations with fiber-optic probes. We will use this system to record the first-pass, and the subsequent-time, site-specific concentrations of test compounds that are labeled with both optical chromophores and radiotracers. Optical measurements will be benchmarked against the assay of the radioactive markers on the same compounds. This will verify the optical method, facilitating use with approved optical contrast agents. Following in-vitro experiments, we will utilize rabbit Vx2 tumors to test the measurement of the first-pass and following kinetics of optical agents and hemoglobin under controlled conditions. We will apply the OP method to estimate microvascular density (peak concentration of optical agent and hemoglobin concentration) and to characterize capillary leak (endothelial transfer rate constant using a tri-compartment kinetic model), two indices of the angiogenic response. We will also test the ability to sense changes in permeability caused by TNF-alpha and following treatment with anti-angiogenesis agents. Such application of the OP method can result in improved management of treatment in humans, especially for superficial lesions in which kinetics are difficult to assess by current imaging methods.